Development and maintenance of rod and cone photoreceptors require coordinately regulated expression of photoreceptor genes. This regulation occurs at multiple levels and requires the action of a network of transcription regulatory proteins. This regulatory network is centered on the cone-rod homeobox protein CRX and incorporates both photoreceptor-specific transcription factors (TFs) and general chromatin modulators. The ultimate goal of our research is to determine how this regulatory protein network interacts with the chromatin of co-expressed target genes to achieve coordinately regulated spatial and temporal expression, providing a better understanding of photoreceptor development and disease. During the last grant period, we discovered that CRX regulates target gene chromatin configurations by recruiting co-activators capable of catalyzing acetylation of histones and organizing intra-gene interactions between regulatory regions. In the new grant period, we will extend our vision of transcriptional regulation to three dimensions: We hypothesize that rod and cone genes are co-regulated as clusters, requiring appropriate higher-order chromosome organization in the nucleus that is mediated by specific TFs and general epigenetic modulators. To test this hypothesis, Aim 1 will determine the role of histone H3 lysine 4 methylation, a positive mark that occurs downstream of acetylation, by assessing the effects of deleting three catalytic enzymes, MLL1, MLL2 and MLL3 in rods, cones and their precursors; Aim 2 will reveal specific intra- and inter-chromosome interactions of co-regulated photoreceptor genes and their co-localization in rod vs. cone nuclei using state-of-the-art epigenetic technologies. To determine if these chromosome organizations contribute to transcription co-regulation during subtype specification and maintenance, profiles of chromosome interactions, TF binding, histone marks and transcription will be studied at various developmental ages. We will also determine if the transcription regulatory network mediates the specific chromosome interactions by analyzing mutant retinas lacking specific TFs or epigenetic modulators. This study will provide the first targeted assessment of transcriptional co-regulation of photoreceptor genes and determine the underlying molecular and cellular mechanisms. The new knowledge gained will have implications in developing stem cell-based cell replacement therapy as well as providing new targets for other therapeutic interventions.